Modern biotechnological techniques require the visualization of very small amounts of biological materials, for instance, nucleic acids.
Present techniques for visualization of nucleic acid fragments make use of either hybridization probes or direct visualization. Probes utilize radioisotopes which are difficult to handle and have the potential of being detrimental to the health of researchers and the environment.
For direct visualization, silver staining is the preferred method for visualizing an image of nucleic acids in gels.
The silver staining methods currently used fall into one of two categories based on the chemical state of the silver ions that prime the staining reaction. Alkaline methods use a diamine complex of silver nitrate in a highly alkaline environment and usually develop the image in dilute acid solutions of formaldehyde. In contrast, acidic methods use silver nitrate solutions for gel impregnation and usually use alkaline developing solutions containing formaldehyde. Reportedly, alkaline methods are less sensitive, but better suited for thicker gels, whereas acidic methods are more rapid, but work best with thin gels.
The above methods are described in Sommerville, L. L., and Wang, K., Biochem. Biophys. Res. Commun., 102:53-58 (1981); Boulikas, T., and Hancock, R. J., Biochem. Biophys. Methods, 5:219-228 (1981); Guillemette, J. G., and Lewis, P. N., Electrophoresis, 4:92-94 (1983); Kolodny, G. M., Anal. Biochem., 138:66-67 (1984); Beidler, J. L., Hilliard, P. R., and Rill, R. L., Anal. Biochem., 126:374-380 (1982); Goldman, D., and Merril, C. R., Electrophoresis, 3:24-26 (1982); Merril, C. R., Harrington, M., and Alley, V., Electrophoresis, 5:289-297 (1984); Blum, H., Beier, H., and Gross, H. J., Electrophoresis, 8:93-99; Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H., Science, 211:1437-1438 (1981); Heukeshoven, J., and Dernick, R., Electrophoresis, 6:103-112 (1985); Nielsen, B. L., and Brown, L. R., Anal. Biochem., 141:311-315 (1984); Allen, R. C., Graves, G., and Budowle, B., Biotechniques, 7:736-744 (1989); Rabilloud, T., Electrophoresis, 11:785-794 (1990); Merril, C. R., Meth. Enzymol., 182:477-488 (1990); Gottlieb, M., and Chavco, K., Anal. Biochem., 165:33-37 (1987); Heukeshoven, J., and Dernick, R., Electrophoresis, 6:103-112 (1985); Wray, W., Boulikas, T., Wray, V. P., and Hancock, R., Anal. Biochem., 118:197-203 (1981) and Switzer, R. C., III, Merril, C. R., and Shifrin, S., Anal. Biochem., 98:231-237 (1979).
Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982) and Ausubel, F. M., et al., Current Protocols in Molecular Biology, Current Protocols (1993), two well-known and standard laboratory reference manuals, disclose silver staining techniques.
A method described in Molecular Cloning at pages 18.56 and 18.57 as suitable for silver staining proteins is that of Sammons, et al., Electrophoresis, 2:135 (1981) with improvements by Schoenle, et al., J. Biol. Chem., 259:12112 (1984). This method does not include formaldehyde in the impregnating solution, as compared with the method of the invention.
A method in Current Protocols in Molecular Biology at page 10.6.5 for silver staining proteins is that of Blum, et al., Electrophoresis, 8:93-99 (1987). Blum, et al. does not include formaldehyde in either the impregnating or developing solution, as compared with the method of the invention.
All patents and publications cited herein are incorporated by reference.
The methods of the prior art all have drawbacks which limit their usefulness, e.g., they are less sensitive, have problems with unspecific background, call for numerous steps, and are cumbersome to perform.
The methods of the prior art have also employed a variety of reducing or oxidizing solutions to pretreat gels after fixation of the proteins or nucleic acids to increase the sensitivity and efficiency of silver staining. The method of the present invention does not require a pretreatment of the gels to maintain sensitivity.